IR dyes for fluorescence imaging

ABSTRACT

A method for organ imaging, comprising: administering to a subject a diagnostic effective amount of 2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-phenoxycyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate or 2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate. In one embodiment, the organ includes one or more of kidney, bladder, liver, gall bladder, spleen, intestine, heart, lungs and muscle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/949,654 filed Nov. 23, 2015, allowed, which application claimspriority to U.S. Provisional Patent Application No. 62/084,971, filedNov. 26, 2014, the teachings of which are hereby incorporated byreference in their entities for all purposes.

FIELD OF THE INVENTION

The present disclosure provides for methods of fluorescence imaging.

BACKGROUND OF THE INVENTION

Conventional laparoscopic and robotic laparoscopic techniques are bothrapidly growing practices in the fields of colorectal and gynecologicsurgery. Recent studies as described in Simorov et al., “Laparoscopiccolon resection trends in utilization and rate of conversion to openprocedure: a national database review of academic medical centers” AnnSurg. 2012 256(3), 462-468; Wright et al., “Robotically assisted vslaparoscopic hysterectomy among women with benign gynecologic disease”JAMA 2013 309(7), 689-698; and, Park et al., “Ureteral injury ingynecologic surgery: a 5-year review in a community hospital” Korean J.Urol. 2012 53(2), 120-125, report 42% of all colorectal resections andgreater than 30% of gynecologic procedures are attempted in this manner.

A laparoscopic approach offers several advantages over traditional openabdominal surgery, including decreased postoperative pain, shorterhospital length of stay, fewer surgical site infections, and decreasedoverall hospital cost, as described in Kiran et al., “Laparoscopicapproach significantly reduces surgical site infections after colorectalsurgery: data from national surgical quality improvement program” J AmColl Surg. 2010 211(2) 232-238; Bilimoria et al., “Laparoscopic-assistedvs. open colectomy for cancer: comparison of short-term outcomes from121 hospitals” J Gastrointest Surg. 2008 12(11) 2001-2009; Juo et al.,“Is Minimally Invasive Colon Resection Better Than TraditionalApproaches?: First Comprehensive National Examination With PropensityScore Matching” JAMA Surg. 2014 149(2) 177-184; Wilson et al.,“Laparoscopic colectomy is associated with a lower incidence ofpostoperative complications compared with open colectomy: a propensityscore-matched cohort analysis” Colorectal Dis. 5 2014 16(5) 382-389;Kobayashi et al., “Total laparoscopic hysterectomy in 1253 patientsusing an early ureteral identification technique” J Obstet Gynaecol Res.2012 38(9) 1194-1200; Makinen et al., “Ten years of progress-improvedhysterectomy outcomes in Finland 1996-2006: a longitudinal observationstudy” BMJ Open 2013 3(10) e003169.

However, limited tactile sensation and two-dimensional images can leadto iatrogenic ureter injury. Although infrequent, laparoscopic ureterinjury remains a serious complication with significant associatedmorbidity. Reports indicate an incidence between 0.1-7.6% for colorectaland gynecologic surgery with more than 80% of these failing to berecognized intraoperatively, as described in Park et al.; Palaniappa etal., “Incidence of iatrogenic ureteral injury after laparoscopiccolectomy” Arch Surg. 2012 147(3), 267-271; and, da Silva et al., “Roleof prophylactic ureteric stents in colorectal surgery” Asian J EndoscSurg. 2012 5(3), 105-110. Current techniques for intraoperative ureteridentification include ureteral stent placement, x-ray fluoroscopy, andvisible dyes; however, both stents and fluoroscopy carry additional riskto the patient, and visible dyes are often not sensitive, as describedin Tanaka et al., “Real-time intraoperative ureteral guidance usinginvisible near-infrared fluorescence” J Urol. 2007 178(5), 2197-2202;Bothwell et al., “Prophylactic ureteral catheterization in colonsurgery. A five-year review” Dis Colon Rectum. 1994 37(4), 330-334; Woodet al., “Routine use of ureteric catheters at laparoscopic hysterectomymay cause unnecessary complications” J Am Assoc Gynecol Laparosc. 19963(3), 393-397; and, Brandes et al., “Diagnosis and management ofureteric injury: an evidence-based analysis” BJU Int. 2004 94(3),277-289.

Fluorescence imaging using cyanine dyes is a rapidly emerging field tosupport surgical navigation and provide real-time illumination ofanatomic structures. Emissions in the 700-900 nm range may avoidinterference from tissue auto-fluorescence and can penetrateapproximately 1 cm of tissue, as described in Adams et al., “Comparisonof visible and near-infrared wavelength-excitable fluorescent dyes formolecular imaging of cancer” J Biomed Opt. 2007 12(2), 024017; and,Keereweer et al., “Optical Image-Guided Cancer Surgery: Challenges andLimitations” Clin Cancer Res. 2013 19(14), 3745-3754.

Another application of fluorescence imaging is for the real-timeintra-operative imaging of the biliary anatomy, including the biliaryduct and cystic duct. Current methods often use indocyanine green (ICG)dye by either intra-biliary injection or intravenous injection beforesurgery. However, studies have shown clear problems in using ICG dye.These include poor efficiency and kinetics of excretion into bile(Tanaka et al., “Real-time intraoperative assessment of the extrahepaticbile ducts in rats and pigs using invisible near-infrared fluorescentlight” Surgery 2008 144(1) 39-48) and adverse reaction with the patient(Benya et al., “Adverse reactions to indocyanine green: a case reportand a review of the literature” Cathet Cardiovasc Diagn. 1989 17(4)231-233).

There exists a need for sensitive compositions and methods to detect andmeasure an internal target non-invasively. Specifically, there exists aneed for improved, stable cyanine dyes to detect injuries to variousorgans that may occur during laparoscopic or robotic surgery. Thepresent invention satisfies these and other needs.

BRIEF SUMMARY OF THE INVENTION

In an embodiment, the present invention includes a method for kidneyureter imaging, comprising: administering to a subject a diagnosticeffective amount of2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonatecompound of Formula 1:

or a solvate or polymorph thereof and having a pharmaceuticallyacceptable cation, wherein the administering is performed at one or moretimes selected from the group consisting of before a procedure, during aprocedure, after a procedure and combinations thereof, exposing tissueof the subject's renal system to electromagnetic radiation; anddetecting fluorescence radiation from the compound.

In one embodiment, the method includes administering the compound ofFormula 1 intravenously.

In one embodiment, the method includes administering the compound ofFormula 1 wherein the pharmaceutically acceptable cation is selectedfrom the group consisting of potassium or sodium.

In one embodiment, the method includes administering the compound ofFormula 1 in combination with a pharmaceutically acceptable carrierselected from the group consisting of physiological sterile salinesolution, sterile water solution, pyrogen-free water solution, isotonicsaline solution, and phosphate buffer solution.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 3000.0 μg/kg and approximately 1500.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 1500.0 μg/kg and approximately 1000.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 1000.0 μg/kg and approximately 500.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 500.0 μg/kg and approximately 170.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 170.0 μg/kg and approximately 120.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 120.0 μg/kg and approximately 60.0 μg/kg.

In one embodiment, the method includes measuring a fluorescenceintensity of the administered compound of Formula 1 remaining at thetissue of the subject's renal system at a time period afteradministering. In certain aspects, the methods provide visualizing thecompound of Formula 1 in urine or bile.

In one embodiment, the method includes the measured fluorescenceintensity of the administered compound of Formula 1 is backgroundfluorescence approximately 24 hours after administering.

In one embodiment, the method includes the procedure selected from thegroup consisting of a laparoscopic procedure, a robotic procedure, arobotic laparoscopic procedure, and an open procedure.

In one embodiment, the method includes the measured fluorescenceintensity of the administered compound of Formula 1 is higher in thekidney as compared to a measured fluorescence intensity of theadministered compound of Formula 1 in one or more of the spleen,intestine, heart, lungs, muscle, or combinations thereof approximatelyup to six hours after administering.

In one embodiment, the present invention provides a solid form (apolymorph), which is Form A of Formula 1. Form A of Formula 1 has anX-ray powder diffraction pattern comprising a peak, in terms of 2-theta,at about 4.3°. Form A has an X-ray powder diffraction pattern comprisinga peak, in terms of 2-theta, at about 4.3°, about 9.6°, about 12.9°,about 18.3°, and about 20.8°. Form A has an X-ray powder diffractionpattern substantially as shown in FIG. 1E.

In one embodiment, the present invention provides a method for biliaryduct imaging, comprising administering a compound of Formula 1 to asubject; and detecting fluorescence radiation from the compound.

In another embodiment, the invention provides a polymorph or solid form(Form A) of the compound of Formula 1 in that it provides an XRPDpattern comprising peaks substantially as set out in Table 2.

In one embodiment, the present invention provides a solid form (apolymorph), which is Form B of Formula 1. Form B of Formula 1 has anX-ray powder diffraction pattern comprising a peak, in terms of 2-theta,at about 21.2°. Form B has an X-ray powder diffraction patterncomprising a peak, in terms of 2-theta, at about 5.3°, about 14.2°,about 14.3°, about 20.7°, and about 21.2°. Form B has an X-ray powderdiffraction pattern substantially as shown in FIG. 1H.

In an embodiment, the invention includes a method for liver biliaryimaging, comprising: administering to a subject a diagnostic effectiveamount of2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-phenoxycyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonateof Formula 2:

or a solvate or polymorph thereof and having a pharmaceuticallyacceptable cation, wherein the administering is performed at one or moretimes selected from the group consisting of before a procedure, during aprocedure, after a procedure and combinations thereof, exposing tissueof the subject's liver system to electromagnetic radiation; anddetecting fluorescence radiation emission from the compound.

In one embodiment, the method includes administering the compound ofFormula 2 intravenously.

In one embodiment, the method includes administering the compound ofFormula 2 wherein the pharmaceutically acceptable cation is selectedfrom the group consisting of potassium or sodium.

In one embodiment, the method includes administering the compound ofFormula 2 in combination with a pharmaceutically acceptable carrierselected from the group consisting of physiological sterile salinesolution, sterile water solution, pyrogen-free water solution, isotonicsaline solution, and phosphate buffer solution.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 3000.0 μg/kg and approximately 1500.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 1500.0 μg/kg and approximately 1000.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 1000.0 μg/kg and approximately 500.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 500.0 μg/kg and approximately 170.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 170.0 μg/kg and approximately 120.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 120.0 μg/kg and approximately 60.0 μg/kg.

In one embodiment, the method includes measuring a fluorescenceintensity of the administered compound of Formula 2 remaining at thetissue of the subject's liver system at a time period afteradministering.

In certain aspects, the methods provide visualizing the compound ofFormula 2 in urine or bile.

In one embodiment, the method includes the measured fluorescenceintensity of the administered compound of Formula 2 is backgroundfluorescence approximately 24 hours after administering.

In one embodiment, the method includes the procedure selected from thegroup consisting of a laparoscopic procedure, a robotic procedure, arobotic laparoscopic procedure, and an open procedure.

In one embodiment, the method includes the measured fluorescenceintensity of the administered compound of Formula 2 is higher in theliver as compared to a measured fluorescence intensity of theadministered compound of Formula 2 in one or more of the spleen,intestine, heart, lungs, muscle, or combinations thereof approximatelyup to six hours after administering.

In an embodiment, the invention includes a method for liver cystic ductimaging, comprising: administering to a subject a diagnostic effectiveamount of2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-phenoxycyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonateof Formula 2:

or a solvate or polymorph thereof and having a pharmaceuticallyacceptable cation, wherein the administering is performed at one or moretimes selected from the group consisting of before a procedure, during aprocedure, after a procedure and combinations thereof, exposing tissueof the subject's liver system to electromagnetic radiation; anddetecting fluorescence radiation from the compound.

In one embodiment, the method includes administering the compound ofFormula 2 intravenously.

In one embodiment, the method includes administering the compound ofFormula 2 wherein the pharmaceutically acceptable cation is selectedfrom the group consisting of potassium or sodium.

In one embodiment, the method includes administering the compound ofFormula 2 in combination with a pharmaceutically acceptable carrierselected from the group consisting of physiological sterile salinesolution, sterile water solution, pyrogen-free water solution, isotonicsaline solution, and phosphate buffer solution.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 3000.0 μg/kg and approximately 1500.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 1500.0 μg/kg and approximately 1000.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 1000.0 μg/kg and approximately 500.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 500.0 μg/kg and approximately 170.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 170.0 μg/kg and approximately 120.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 120.0 μg/kg and approximately 60.0 μg/kg.

In one embodiment, the method includes measuring a fluorescenceintensity of the administered compound of Formula 2 remaining at thetissue of the subject's liver system at a time period afteradministering.

In one embodiment, the method includes the measured fluorescenceintensity of the administered compound of Formula 2 is backgroundfluorescence approximately 24 hours after administering.

In one embodiment, the method includes the procedure selected from thegroup consisting of a laparoscopic procedure, a robotic procedure, arobotic laparoscopic procedure, and an open procedure.

In one embodiment, the present invention provides a method for kidneyureter imaging, comprising administering a compound of Formula 2 to asubject; and detecting fluorescence radiation from the compound.

In one embodiment, the method includes the measured fluorescenceintensity of the administered compound of Formula 2 is higher in theliver as compared to a measured fluorescence intensity of theadministered compound of Formula 2 in one or more of the spleen,intestine, heart, lungs, muscle, or combinations thereof approximatelyup to six hours after administering.

In one embodiment, a pharmaceutical composition comprises a diagnosticimaging amount of a compound of Formula 1, a pharmaceutically acceptablecation, and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition comprising adiagnostic imaging amount of a compound of Formula 1, a pharmaceuticallyacceptable cation, and a pharmaceutically acceptable carrier iscontained in an intravenous (IV) bag.

In one embodiment, a pharmaceutical composition comprises a diagnosticimaging amount of a compound of Formula 2, a pharmaceutically acceptablecation, and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition comprising adiagnostic imaging amount of a compound of Formula 2, a pharmaceuticallyacceptable cation, and a pharmaceutically acceptable carrier iscontained in an intravenous (IV) bag.

In one embodiment, the invention includes a kit containing thepharmaceutical composition comprising a diagnostic imaging amount of acompound of Formula 1, a pharmaceutically acceptable cation, and apharmaceutically acceptable carrier, and an instruction manual orinstructions for use. The pharmaceutical composition can be contained inan intravenous (IV) bag.

In other embodiments, the invention includes a kit containing thepharmaceutical composition comprising a diagnostic imaging amount of acompound of Formula 2, a pharmaceutically acceptable cation, and apharmaceutically acceptable carrier, and an instruction manual orinstructions for use. The pharmaceutical composition can be contained inan intravenous (IV) bag.

In an embodiment, the invention includes a composition of mattercomprising:2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-phenoxycyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonateof Formula 2:

or a solvate or polymorph thereof and having a pharmaceuticallyacceptable cation.

Other embodiments, aspects, and objects will become better understoodwhen read with the detailed description and drawings which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

In each of the drawings 800CW corresponds to the compound set forth inComparative example 1; 800BK-sulfonate refers to compound of Formula 1;800BK-NOS refers to compound of Formula 2.

FIGS. 1A-1I show X-ray powder diffraction (XRPD) patterns forvarioussamples. Samples shown in FIGS. 1A and 1C represent Form A. FIG. 1B isForm A, but less crystalline. FIGS. 1D and 1E show the XRPD pattern ofcrystalline Form A. FIG. 1F shows the XRPD of the amorphous form. FIG.1H and FIG. 1I show the XRPD pattern of crystalline Form B. Forcomparison, FIG. 1G is identical to FIG. 1D.

FIGS. 2A, and 2B illustrate in vitro cell-based analyses for A549 cells(A) and A431 cells (B). FIG. 2C shows fluorescence as a function ofconcentration of each dye prepared for in vivo imaging (0.03-1 μM).

FIGS. 3A-C illustrates dorsal view fluorescence images of mice for thecompound of comparative example 1 (FIG. 3A), the compound of Formula 1(FIG. 3B), and the compound of Formula 2 (FIG. 3C) probes. Images arepresented for the approximate time points post probe administration: 30min, 2 hrs, 5 hrs, and 24 hrs. A Pearl® Impulse small animal imagingsystem was used for all animal and organ image acquisitions

FIGS. 4A-C illustrate ventral view fluorescence images of the same micefor the compound of comparative example 1 (FIG. 4A); the compound ofFormula 1 (FIG. 4B), and the compound of Formula 2 probes (FIG. 4C).Images are presented for the approximate time points post probeadministration: 30 min, 2 hrs, 5 hrs, and 24 hrs. Arrows pinpointbladder regional bladder signal. White arrows point to gall bladdersignal.

FIG. 5 illustrates whole body fluorescence intensity numbers for theimages of FIG. 4 for the compound of comparative example 1, the compoundof Formula 1, and the compound of Formula 2 probes.

FIGS. 6A-D show fluorescence data for mouse organs for the compound ofcomparative example 1 (FIG. 6A), the compound of Formula 2 (FIG. 6B),the compound of Formula 1 (FIG. 6C): liver, spleen, intestine, brain,heart/lung, kidney, brain, and muscle (FIG. 6D). Fluorescence signal(800 nm) adjusted to area (pixel) for all probes.

FIG. 7 illustrates image captured of ureters, kidneys and bladder inmouse after administration of the compound of Formula 1 probe.

FIGS. 8A-B illustrate fluorescence detection of probes in a histogram ofstained electrophoresis gels of selected proteins showing non-specificor affinity of the named dyes to human (H), bovine (B), ovalbumin (O),or 5% FBS (fetal bovine serum) (FIG. 8A). In the fluorescence images ofstained electrophoresis gels dark grey represents the signal from thecompound being examined, light grey represents a Coomassie Blueassessment of protein loading and white represents where both colorsreside (FIG. 8B).

FIG. 9 illustrates fluorescent signal (800 nm) analysis of mouse urinefor a control sample (no probe), the compound of Formula 1, the compoundof Formula 2, and indocyanine green (ICG) dye.

FIGS. 10A, 10B, 10C, 10D, and 10E illustrate biliary tract imaging for:50 nmole of ICG in phosphate buffer saline (PBS) solution (FIG. 10A), 1nmole of the compound of Formula 2 in PBS solution (FIG. 10B), 0.5 nmoleof the compound of Formula 2 in PBS solution (FIG. 10C), and 0.1 nmoleof the compound of Formula 2 in PBS solution (FIGS. 10D and 10E). Theimaging areas displayed for each condition were recorded at 1 min, 30min, 2 hrs, 5 hrs, and 24 hrs., respectively (left to right).

FIGS. 11A-11H show images of various mouse organs after administration.Fluorescence images of various mouse organs at 24 and 72 hrspost-administration for the comparative control (FIG. 11A), compound ofFormula 1 (FIGS. 11C and 11E) and compound of Formula 2 (FIGS. 11B and11D). Organs examined included: heart (Ht), lungs (Ln), kidney (Kd),liver (Lv), spleen (Spl), intestine (Int), brain (Br), and muscle (Ms).At 24 hrs, there remains some signal in the liver and kidney but by 72hrs the signal is diminished substantially. FIGS. 11F-11H showintraoperative fluorescence (800 nm, compound of Formula 2) (FIG. 11G),white light (FIG. 11F), and composite images of the liver, gall bladder,and bile duct for compound of Formula 2 (0.1 nmole) (FIG. 11H).

FIG. 12 shows a spot test of the injected dye solutions-800CW,800BK-sulfonate (compound of Formula 1; 800BK or BK) and 800BK-NOS(compound of Formula 2; 800NOS or NOS).

FIGS. 13A-13C show whole animal imaging of dorsal and ventral view of arepresentative mouse injected with one of the dyes tested. Each panel offigures shows a dorsal and ventral view of the animal at 15 minutes, 1hour, 2 hours, 4 hours 6 hours and 24 hours after injection. An animalinjected with 800CW dye, 800NOS dye, and 800BK dye are shown in FIG.13A, FIG. 13B and FIG. 13C, respectively.

FIG. 14 shows the whole animal LUT scale, the organ 1/10^(th) LUT scale,and the organ 1/100^(th) LUT scale.

FIGS. 15A-15D show the signal intensity of the kidney (lower leftcorner), liver (lower right corner), lungs (upper left corner) andmuscle (upper right corner) on the organ 1/10^(th) LUT scale. Organsfrom an animal injected with no probe (control), 800CW dye, 800NOS dye,and 800BK dye are shown in FIG. 15A, FIG. 15B, FIG. 15C, and FIG. 15D,respectively.

FIGS. 16A-16D show the signal intensity of the kidney (lower leftcorner), liver (lower right corner), lungs (upper left corner) andmuscle (upper right corner) on the organ 1/100^(th) LUT scale. Organsfrom an animal injected with no probe (control), 800CW dye, 800NOS dye,and 800BK dye are shown in FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D,respectively.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forexample, an embodiment of a method of imaging that comprises using acompound set forth herein would include an aspect in which the methodcomprises using two or more compounds set forth herein.

The term “approximately” or “about” as used herein to modify a numericalvalue indicates a defined range around that value. If “X” were thevalue, “approximately X” or “about X” would indicate a value from 0.9Xto 1.1X, and more preferably, a value from 0.95X to 1.05X. Any referenceto “approximately X” or “about X” specifically indicates at least thevalues X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X,and 1.05X. For example, “approximately X” or “about X” is intended toteach and provide written description support for a claim limitation of,e.g., “0.98X.”

When the quantity “X” only allows whole-integer values (e.g., “Xcarbons”) and X is at most 15, “about X” indicates from (X−1) to (X+1).In this case, “about X” as used herein specifically indicates at leastthe values X, X−1, and X+1. If X is at least 16, the values of 0.90X and1.10X are rounded to the nearest whole-integer values to define theboundaries of the range.

When the modifier “approximately” or “about” is applied to describe thebeginning of a numerical range, it applies to both ends of the range.Thus, “from approximately 700 to 850 nm” is equivalent to “fromapproximately 700 nm to approximately 850 nm.” Thus, “from about 700 to850 nm” is equivalent to “from about 700 nm to about 850 nm.” When“approximately” or “about” is applied to describe the first value of aset of values, it applies to all values in that set. Thus, “about 680,700, or 750 nm” is equivalent to “about 680 nm, about 700 nm, or about750 nm.”

“Balanced charge” as used herein includes the condition that the netcharge for a compound and its associated counterions be zero understandard physiological conditions. In order to achieve a balancedcharge, a skilled person will understand that after the first additionalsulfonato group that balances the +1 charge of the indolinium ring, acationic counterion (e.g., the cation of a Group I metal such as sodium)must be added to balance the negative charge from additional sulfonatogroups. Similarly, anionic counterions must be added to balance anyadditional cationic groups (e.g., most basic amino groups underphysiological conditions).

II. Embodiments

While preferred embodiments of the invention are shown and describedherein, such embodiments are provided by way of example only and are notintended to otherwise limit the scope of the invention. Variousalternatives to the described embodiments of the invention may beemployed in practicing the invention.

Some advantages of cyanine dyes include: (1) cyanine dyes stronglyabsorb and fluoresce light; (2) many cyanine dyes do not rapidlyphoto-bleach under the fluorescence microscope; (3) many structures andsynthetic procedures are available and the class of dyes is versatile;and (4) cyanine dyes are relatively small (a typical molecular weight isabout 1,000 daltons) so they do not cause appreciable stericinterference.

Generally, cyanine dyes are prepared according to the procedures taughtin Hamer, F. M., Cyanine Dyes and Related Compounds, Weissberger, Mass.,ed. Wiley Interscience, N.Y. 1964. For example, U.S. Pat. Nos.6,663,847; 6,887,854; 6,995,274; 7,504,089; 7,547,721; 7,597,878 and8,303,936, incorporated herein by reference, describe synthesismechanisms for a variety of cyanine dyes.

Other cyanine dyes are known which contain reactive functional groups.For example, U.S. Pat. Nos. 4,337,063; 4,404,289 and 4,405,711,incorporated herein by reference, describe a synthesis for a variety ofcyanine dyes having N-hydroxysuccinimide active ester groups. U.S. Pat.No. 4,981,977, incorporated herein by reference, describes a synthesisfor cyanine dyes having carboxylic acid groups. U.S. Pat. No. 5,268,486,incorporated herein by reference, discloses a method for makingarylsulfonate cyanine dyes. U.S. Pat. No. 6,027,709, incorporated hereinby reference, discloses methods for making cyanine dyes havingphosphoramidite groups. U.S. Pat. No. 6,048,982, incorporated herein byreference, discloses methods for making cyanine dyes having a reactivegroup selected from the group consisting of isothiocyanate, isocyanate,phosphoramidite, monochlorotriazine, dichlorotriazine, mono- ordi-halogen substituted pyridine, mono- or di-halogen substituteddiazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimideester, hydroxy sulfosuccinimide ester, imido ester, glyoxal andaldehyde.

As described herein, the present invention provides for the use of acyanine dye of the compound having Formula 1:

or a solvate or polymorph thereof and having a pharmaceuticallyacceptable cation.

In one embodiment, the present invention provides a solid form (apolymorph), which is Form A of Formula 1. Form A of Formula 1 has anX-ray powder diffraction pattern comprising a peak, in terms of 2-theta,at about 4.30. Form A has an X-ray powder diffraction pattern comprisinga peak, in terms of 2-theta, at about 4.3°, about 9.6°, about 12.9°,about 18.3°, and about 20.8°. Form A has an X-ray powder diffractionpattern substantially as shown in FIG. 1E.

In one embodiment, the present invention provides a solid form (apolymorph), which is Form B of Formula 1. Form B of Formula 1 has anX-ray powder diffraction pattern comprising a peak, in terms of 2-theta,at about 21.2°. Form B has an X-ray powder diffraction patterncomprising a peak, in terms of 2-theta, at about 5.3°, about 14.2°,about 14.3°, about 20.7° and about 21.2°. Form B has an X-ray powderdiffraction pattern substantially as shown in FIG. 1H.

In certain aspects, the polymorphs of the invention are formulated intoa composition prior to administration to a subject.

The compounds of Formula 1 can exist in crystalline or noncrystallineform, or as a mixture thereof. For salts of the invention that are incrystalline form, the skilled artisan will appreciate thatpharmaceutically acceptable solvates may be formed wherein solventmolecules are incorporated into the crystalline lattice duringcrystallization. Solvates may involve nonaqueous solvents such asethanol, isopropanol, DMSO, acetic acid, ethanolamine, and ethylacetate.In one embodiment, the invention provides the sodium salt of thecompound of Formula 1 incorporated into the crystalline lattice.

In one aspect, the present invention provides a polymorph of thecompound of Formula 1 in isolated or pure form. “Isolated” or “pure” or“substantially pure” form refers to a sample in which the polymorph ispresent in an amount of >75%,particularly >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%,or >99%, relative to other materials which may be present in the sample.

The polymorphs made according to the methods of the invention can becharacterized by any methodology according to the art. For example, thepolymorphs made according to the methods of the invention may becharacterized by X-ray powder diffraction (XRPD), differential scanningcalorimetry (DSC), thermogravimetric analysis (TGA), hot-stagemicroscopy, and spectroscopy (e.g., Raman, solid state nuclear magneticresonance (ssNMR), and infrared (IR)).

XRPD

Polymorphs according to the invention may be characterized by X-raypowder diffraction patterns (XRPD). The relative intensities of XRPDpeaks can vary, depending upon the sample preparation technique, thesample mounting procedure and the particular instrument employed.Moreover, instrument variation and other factors can affect the2θvalues. Therefore, the XRPD peak assignments can vary by plus or minusabout 0.2 degrees.

The polymorph forms of the invention are useful in the production ofimaging agents and can be obtained by means of a crystallization processto produce crystalline and semi-crystalline forms. In variousembodiments, the crystallization is carried out by either generating thecompound of Formula 1 in a reaction mixture and isolating the desiredpolymorph from the reaction mixture, or by dissolving raw compound in asolvent, optionally with heat, followed by crystallizing/solidifying theproduct by cooling (including active cooling) and/or by the addition ofan antisolvent for a period of time. The crystallization can be followedby drying carried out under controlled conditions until the desiredwater content is reached in the end polymorphic form.

In another embodiment, the present invention provides a method of makinga polymorph of a compound of Formula 1 (Form A or Form B). In variousembodiments, the invention is directed to methods of making a polymorphof the compound of Formula 1, wherein the method involves converting anamorphous form into a desired polymorph. In certain embodiments, themethods comprise exposing a composition comprising an amorphous form toconditions sufficient to convert at least about 50% of the total amountof the amorphous form into at least about 50% of the desired polymorph,and isolating the desired polymorph as needed.

In certain instances, a crystalline solid will be more amenable topurification than an amorphous solid and the crystalline form is able tobe made in higher purity. This is because, under proper conditions, theformation of the crystals tends to exclude impurities from the solid, incontrast to amorphous solids formed in a less controlled manner.Similarly, a crystalline solid will often have better stability than anamorphous solid, as the crystal packing gives a protective effect. Formaterials which are polymorphic, some crystal forms will normally bemore effective than others at excluding impurities and enhancingstability.

In one embodiment, the present invention provides for a compositionaccording to Formula 2:

or a hydrate or polymorph thereof and having a pharmaceuticallyacceptable cation.

One embodiment of the invention includes a method for organ imaging,comprising: administering to a subject a diagnostic effective amount of2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonatecompound of Formula 1:

or a solvate or polymorph thereof and having a pharmaceuticallyacceptable cation, wherein the administering is performed at one or moretimes selected from the group consisting of before a procedure, during aprocedure, after a procedure and combinations thereof, exposing tissueof the subject's organ system to electromagnetic radiation; anddetecting fluorescence radiation from the compound. In one embodiment,the organ includes one or more of kidney, bladder, liver, spleen,intestine, heart, lungs and muscle. In one embodiment, the organ iskidney, bladder or combinations of. In another embodiment, the organ isthe ureter of a kidney.

In one embodiment, the method includes administering the compound ofFormula 1 intravenously. The compound of Formula 1 can be administeredas a bolus injection, e.g., an intravenous bolus injection. In someembodiments, about 5 mL to about 10 mL of a composition comprising thecompound of Formula 1 is administered in a bolus injection.

In one embodiment, the method includes administering the compound ofFormula 1 wherein the pharmaceutically acceptable cation is selectedfrom the group consisting of potassium or sodium.

In one embodiment, the method includes administering the compound ofFormula 1 in combination with a pharmaceutically acceptable carrierselected from the group consisting of physiological sterile salinesolution, sterile water solution, pyrogen-free water solution, isotonicsaline solution, and phosphate buffer solution.

The compound of Formula 1 can be highly soluble in water. In someembodiments, the compound of Formula 1 is re-suspended in water to aconcentration of at least 200 mg/mL, or about 300 mg/mL to about 320mg/mL.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 3000.0 μg/kg and approximately 1500.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 1500.0 μg/kg and approximately 1000.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 1000.0 μg/kg and approximately 500.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 500.0 μg/kg and approximately 170.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 170.0 rig/kg and approximately 120.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 120.0 μg/kg and approximately 60.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 1 at a diagnostic effective amount of the compound rangingbetween approximately 30.0 μg/kg and approximately 500.0 μg/kg.

The compounds of Formula 1 can be non-toxic. They can absorb andfluoresce light, and do not rapidly photo-bleach under fluorescenceimaging. Upon administration, the compounds of Formula 1 can betransported to tissues and organs of the subject via the natural flow ofbodily fluids in the subject. As such, compounds of Formula 1 can becarried or transferred from the site of administration to the desiredsites, tissues and organs for, e.g., visualization.

In certain embodiments, the compounds and methods herein can image thebiliary tract which includes any part of the liver, gall bladder,spleen, small intestine, and associated ducts. In certain instances, thebiliary tract includes the intrahepatic bile ducts, cysticduct-gallbladder to common bile duct—and common bile duct—liver andgallbladder to small intestine. In some instances, the compounds hereinare found in a subject's bile or urine at a time period afteradministering. The present invention provides compositions of thecompounds of Formula 1 or 2 in urine or bile.

In certain embodiments, the methods herein provide visualizing thecompounds of Formula 1 or 2 in urine or bile.

In one embodiment, the method includes measuring a fluorescenceintensity of the administered compound of Formula 1 remaining at thetissue of the subject's organ at a time period after administering. Inone embodiment, the organ includes one or more of kidney, bladder,liver, gall bladder, spleen, intestine, heart, lungs and muscle. In oneembodiment, the organ is kidney, bladder or combinations. In anotherembodiment, the organ is the ureter of a kidney. In some embodiments,the method includes measuring a fluorescence intensity of theadministered compound of Formula 1 remaining in the subject's urine orbile at a time period after administering.

In one embodiment, the method includes the measured fluorescenceintensity of the administered compound of Formula 1 is backgroundfluorescence approximately 24 hours after administering.

In one embodiment, the method includes the procedure selected from thegroup consisting of a laparoscopic procedure, a robotic procedure, arobotic laparoscopic procedure, an endoscopic procedure, and an openprocedure.

In one embodiment, the method includes the measured fluorescenceintensity of the administered compound of Formula 1 is higher in thekidney as compared to a measured fluorescence intensity of theadministered compound of Formula 1 in one or more of the spleen,intestine, heart, lungs, muscle, or combinations thereof approximatelyup to six hours after administering.

In one embodiment, a pharmaceutical composition comprises a diagnosticimaging amount of a compound of Formula 1, a pharmaceutically acceptablecation, and a pharmaceutically acceptable carrier.

In another embodiment, a method for organ imaging, comprising:administering to a subject a diagnostic effective amount of2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-phenoxycyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonateof Formula 2:

or a solvate or polymorph thereof and having a pharmaceuticallyacceptable cation, wherein the administering is performed at one or moretimes selected from the group consisting of before a procedure, during aprocedure, after a procedure and combinations thereof, exposing tissueof the subject's organ system to electromagnetic radiation; anddetecting fluorescence radiation from the compound. In one embodiment,the organ includes one or more of kidney, bladder, liver, spleen,intestine, heart, lungs and muscle. In one embodiment, the organ is aliver. In one embodiment, the organ is the biliary duct of a liver. Inone embodiment, the organ is the cystic duct of a liver.

In one embodiment, the method includes administering the compound ofFormula 2 intravenously. The compound of Formula 2 can be administeredas a bolus injection, e.g., an intravenous bolus injection. In someembodiments, about 5 mL to about 10 mL of a composition comprising thecompound of Formula 2 is administered in a bolus injection.

In one embodiment, the method includes administering the compound ofFormula 2 wherein the pharmaceutically acceptable cation is selectedfrom the group consisting of potassium or sodium.

In one embodiment, the method includes administering the compound ofFormula 2 in combination with a pharmaceutically acceptable carrierselected from the group consisting of physiological sterile salinesolution, sterile water solution, pyrogen-free water solution, isotonicsaline solution, and phosphate buffer solution.

The compound of Formula 2 can be highly soluble in water. In someembodiments, the compound of Formula 2 is resuspended in water to aconcentration of at least 200 mg/mL, about 300 mg/mL or about 320 mg/mL.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 3000.0 μg/kg and approximately 1500.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 1500.0 μg/kg and approximately 1000.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 1000.0 μg/kg and approximately 500.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 500.0 μg/kg and approximately 170.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 170.0 μg/kg and approximately 120.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 120.0 μg/kg and approximately 60.0 μg/kg.

In one embodiment, the method includes administering the compound ofFormula 2 at a diagnostic effective amount of the compound rangingbetween approximately 30.0 μg/kg and approximately 500.0 μg/kg.

The compounds of Formula 2 can be non-toxic. They can absorb andfluoresce light, and do not rapidly photo-bleach under fluorescenceimaging. Upon administration, the compounds of Formula 2 can betransported to tissues and organs of the subject via the natural flow ofbodily fluids in the subject. As such, compounds of Formula 2 can becarried or transferred from the site of administration to the desiredsites, tissues and organs for, e.g., visualization.

In one embodiment, the method includes measuring a fluorescenceintensity of the administered compound of Formula 2 remaining at thetissue of the subject's organ system at a time period afteradministering. In one embodiment, the organ includes one or more ofkidney, bladder, liver, gall bladder, spleen, intestine, heart, lungsand muscle. In one embodiment, the organ is the liver. In anotherembodiment, the organ is the biliary duct of a liver. In anotherembodiment, the organ is the cystic duct of a liver. In someembodiments, the method includes measuring a fluorescence intensity ofthe administered compound of Formula 2 remaining in the subject's urineor bile at a time period after administering.

In one embodiment, the method includes the measured fluorescenceintensity of the administered compound of Formula 2 is backgroundfluorescence approximately 24 hours after administering.

In one embodiment, the method includes the procedure selected from thegroup consisting of a laparoscopic procedure, a robotic procedure, arobotic laparoscopic procedure, an endoscopic procedure, and an openprocedure.

In one embodiment, the method includes the measured fluorescenceintensity of the administered compound of Formula 2 is higher in theliver as compared to a measured fluorescence intensity of theadministered compound of Formula 2 in one or more of the spleen,intestine, heart, lungs, muscle, or combinations thereof approximatelyup to six hours after administering.

In one embodiment, a pharmaceutical composition comprises a diagnosticimaging amount of a compound of Formula 2, a pharmaceutically acceptablecation, and a pharmaceutically acceptable carrier.

In some embodiments, a pharmaceutical composition of compound of Formula1 or compound of Formula 2 are in combination with a pharmaceuticallyacceptable cation and a pharmaceutically acceptable carrier in adiagnostic effective amount. In such embodiments, the pharmaceuticalcomposition is contained in an intravenous drip bag.

In selected embodiments, the diagnostic effective amount of each of thecompound of Formula 1 and the compound of Formula 2, is independentlyless than, for example, 3000.0, 2800.0, 2600.0, 2400.0, 2200.0, 2000.0,1800.0, 1600.0, 1400.0, 1200.0, 1000.0, 950.0, 900.0, 850.0, 800.0,750.0, 700.0, 650.0, 600.0, 550.0, 500.0, 490.0, 480.0, 470.0, 460.0,450.0, 440.0, 430.0, 420.0, 410.0, 400.0, 390.0, 380.0, 370.0, 360.0,350.0, 340.0, 330.0, 320.0, 310.0, 300.0, 290.0, 280.0, 270.0, 260.0,250.0, 240.0, 230.0, 220.0, 210.0, 200.0, 190.0, 180.0, 170.0, 160.0,150.0, 140.0, 130.0, 120.0, 110.0, 100.0, 95.0, 90.0, 85.0, 80.0, 75.0,70.0, 65.0, 60.0, 55.0, 50.0, 49.0, 48.0, 47.0, 46.0, 45.0, 44.0, 43.0,42.0, 41.0, 40.0, 39.5, 39.0, 38.5, 38.0, 37.5, 37.0, 36.5, 36.0, 35.5,35.0, 34.5, 34.0, 33.5, 33.0, 32.5, 32.0, 31.5, 31.0, 30.9, 30.8, 30.7,30.6, 30.5, 30.4, 30.3, 30.2, 30.1 μg/kg.

In selected embodiments, the diagnostic effective amount of each of thecompound of Formula 1 and the compound of Formula 2, is independentlygreater than, for example, 2800.0, 2600.0, 2400.0, 2200.0, 2000.0,1800.0, 1600.0, 1400.0, 1200.0, 1000.0, 950.0, 900.0, 850.0, 800.0,750.0, 700.0, 650.0, 600.0, 550.0, 500.0, 490.0, 480.0, 470.0, 460.0,450.0, 440.0, 430.0, 420.0, 410.0, 400.0, 390.0, 380.0, 370.0, 360.0,350.0, 340.0, 330.0, 320.0, 310.0, 300.0, 290.0, 280.0, 270.0, 260.0,250.0, 240.0, 230.0, 220.0, 210.0, 200.0, 190.0, 180.0, 170.0, 160.0,150.0, 140.0, 130.0, 120.0, 110.0, 100.0, 95.0, 90.0, 85.0, 80.0, 75.0,70.0, 65.0, 60.0, 55.0, 50.0, 49.0, 48.0, 47.0, 46.0, 45.0, 44.0, 43.0,42.0, 41.0, 40.0, 39.5, 39.0, 38.5, 38.0, 37.5, 37.0, 36.5, 36.0, 35.5,35.0, 34.5, 34.0, 33.5, 33.0, 32.5, 32.0, 31.5, 31.0, 30.9, 30.8, 30.7,30.6, 30.5, 30.4, 30.3, 30.2, 30.1, 30.0 μg/kg.

In selected embodiments, the diagnostic effective amount of each of thecompound of Formula 1 and the compound of Formula 2, is independently inthe range from approximately 30.0 μg/kg to approximately 3000.0 μg/kg,approximately 30.1 μg/kg to approximately 2800.0 jg/kg, approximately30.2 μg/kg to approximately 2600.0 μg/kg, approximately 30.3 μg/kg toapproximately 2400.0 μg/kg, approximately 30.4 μg/kg to approximately2200.0 μg/kg, approximately 30.5 μg/kg to approximately 2000.0 μg/kg,approximately 30.6 μg/kg to approximately 1800.0 μg/kg, approximately30.7 μg/kg to approximately 1600.0 μg/kg, approximately 30.8 μg/kg toapproximately 1400.0 μg/kg, approximately 30.9 μg/kg to approximately1200.0 μg/kg, approximately 31.0 μg/kg to approximately 1000.0 μg/kg,approximately 31.5 μg/kg to approximately 950.0 μg/kg, approximately32.0 μg/kg to approximately 900.0 μg/kg, approximately 32.5 μg/kg toapproximately 850.0 μg/kg, approximately 33.0 μg/kg to approximately800.0 μg/kg, approximately 33.5 μg/kg to approximately 750.0 μg/kg,approximately 34.0 μg/kg to approximately 700.0 μg/kg, approximately34.5 μg/kg to approximately 650.0 μg/kg, approximately 35.0 μg/kg toapproximately 600.0 μg/kg, approximately 35.5 μg/kg to approximately550.0 μg/kg, approximately 36.0 μg/kg to approximately 500.0 μg/kg,approximately 36.5 μg/kg to approximately 490.0 μg/kg, approximately37.0 μg/kg to approximately 480.0 μg/kg, approximately 37.5 μg/kg toapproximately 470.0 μg/kg, approximately 38.0 μg/kg to approximately460.0 μg/kg, approximately 38.5 μg/kg to approximately 450.0 μg/kg,approximately 39.0 μg/kg to approximately 440.0 μg/kg, approximately39.5 μg/kg to approximately 430.0 μg/kg, approximately 40.0 μg/kg toapproximately 420.0 μg/kg, approximately 41.0 μg/kg to approximately410.0 μg/kg, approximately 42.0 μg/kg to approximately 400.0 μg/kg,approximately 43.0 μg/kg to approximately 390.0 μg/kg, approximately44.0 μg/kg to approximately 380.0 μg/kg, approximately 45.0 μg/kg toapproximately 370.0 μg/kg, approximately 46.0 μg/kg to approximately360.0 μg/kg, approximately 47.0 μg/kg to approximately 350.0 μg/kg,approximately 48.0 μg/kg to approximately 340.0 μg/kg, approximately49.0 μg/kg to approximately 330.0 μg/kg, approximately 50.0 μg/kg toapproximately 320.0 μg/kg, approximately 55.0 μg/kg to approximately310.0 μg/kg, approximately 60.0 μg/kg to approximately 300.0 μg/kg,approximately 65.0 μg/kg to approximately 290.0 μg/kg, approximately70.0 μg/kg to approximately 280.0 μg/kg, approximately 75.0 μg/kg toapproximately 270.0 μg/kg, approximately 80.0 μg/kg to approximately260.0 μg/kg, approximately 85.0 μg/kg to approximately 250.0 μg/kg,approximately 90.0 μg/kg to approximately 240.0 μg/kg, approximately95.0 μg/kg to approximately 230.0 μg/kg, approximately 100.0 μg/kg toapproximately 220.0 μg/kg, approximately 110.0 μg/kg to approximately210.0 μg/kg, approximately 120.0 μg/kg to approximately 200.0 μg/kg,approximately 130.0 μg/kg to approximately 190.0 μg/kg, approximately140.0 μg/kg to approximately 180.0 μg/kg, and approximately 150.0 μg/kgto approximately 170.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 1 is in the range from approximately 3000.0 μg/kg toapproximately 1500.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 1 is in the range from approximately 1500.0 μg/kg toapproximately 1000.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 1 is in the range from approximately 1000.0 μg/kg toapproximately 500.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 1 is in the range from approximately 500.0 μg/kg toapproximately 170.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 1 is in the range from approximately 170.0 μg/kg toapproximately 120.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 1 is in the range from approximately 120.0 μg/kg toapproximately 60.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 1 is in the range from approximately 30.0 μg/kg to approximately500.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 2 is in the range from approximately 3000.0 μg/kg toapproximately 1500.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 2 is in the range from approximately 1500.0 μg/kg toapproximately 1000.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 2 is in the range from approximately 1000.0 μg/kg toapproximately 500.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 2 is in the range from approximately 500.0 μg/kg toapproximately 170.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 2 is in the range from approximately 170.0 μg/kg toapproximately 120.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 2 is in the range from approximately 120.0 μg/kg toapproximately 60.0 μg/kg.

In one embodiment, the diagnostic effective amount of the compound ofFormula 2 is in the range from approximately 30.0 μg/kg to approximately500.0 μg/kg.

The diagnostic effective amount of each of the compound of Formula 1 andthe compound of Formula 2 according to the invention is effective over awide dosage range. The exact dosage will depend upon the route ofadministration, the form in which each compound is administered, thegender, age, and body weight of the subject to be treated, and thepreference and experience of the administrator.

In selected embodiments, the fluorescence intensity of each of theadministered compound of Formula 1 and the administered compound ofFormula 2 may be independently measured at a time period of, forexample, less than 24 hrs, 23 hrs, 22 hrs, 21 hrs, 20 hrs, 19 hrs, 18hrs, 17 hrs, 16 hrs, 15 hrs, 14 hrs, 13 hrs, 12 hrs, 11 hrs, 10 hrs, 9.5hrs, 9.0 hrs, 8.5 hrs, 8.0 hrs, 7.5 hrs, 7.0 hrs, 6.5 hrs, 6.0 hrs, 5.5hrs, 5.0 hrs, 4.75 hrs, 4.50 hrs, 4.25 hrs, 4.00 hrs, 3.75 hrs, 3.50hrs, 3.25 hrs, 3.00 hrs, 2.75 hrs, 2.50 hrs, 2.25 hrs, 2.00 hrs, 1.75hrs, 1.50 hrs, 1.25 hrs, 1.00 hrs, 0.90 hrs, 0.80 hrs, 0.70 hrs, 0.60hrs, 0.50 hrs, 0.40 hrs, 0.30 hrs, 0.20 hrs after administering.

In selected embodiments, the fluorescence intensity of each of theadministered compound of Formula 1 and the administered compound ofFormula 2 may be independently measured at a time period of, forexample, greater than 23 hrs, 22 hrs, 21 hrs, 20 hrs, 19 hrs, 18 hrs, 17hrs, 16 hrs, 15 hrs, 14 hrs, 13 hrs, 12 hrs, 11 hrs, 10 hrs, 9.5 hrs,9.0 hrs, 8.5 hrs, 8.0 hrs, 7.5 hrs, 7.0 hrs, 6.5 hrs, 6.0 hrs, 5.5 hrs,5.0 hrs, 4.75 hrs, 4.50 hrs, 4.25 hrs, 4.00 hrs, 3.75 hrs, 3.50 hrs,3.25 hrs, 3.00 hrs, 2.75 hrs, 2.50 hrs, 2.25 hrs, 2.00 hrs, 1.75 hrs,1.50 hrs, 1.25 hrs, 1.00 hrs, 0.90 hrs, 0.80 hrs, 0.70 hrs, 0.60 hrs,0.50 hrs, 0.40 hrs, 0.30 hrs, 0.20 hrs, 0.10 hrs after administering.

In selected embodiments, the fluorescence intensity of each of theadministered compound of Formula 1 and the administered compound ofFormula 2 may be independently measured at a time period in the rangefrom approximately 0.10 hrs to approximately 24 hrs, approximately 0.20hrs to approximately 23 hrs, approximately 0.30 hrs to approximately 22hrs, approximately 0.40 hrs to approximately 21 hrs, approximately 0.50hrs to approximately 20 hrs, approximately 0.60 hrs to approximately 19hrs, approximately 0.70 hrs to approximately 18 hrs, approximately 0.80hrs to approximately 17 hrs, approximately 0.90 hrs to approximately 16hrs, approximately 1.00 hr to approximately 15 hrs, approximately 1.25hrs to approximately 14 hrs, approximately 1.50 hrs to approximately 13hrs, approximately 1.75 hrs to approximately 12 hrs, approximately 2.00hrs to approximately 11 hrs, approximately 2.25 hrs to approximately 10hrs, approximately 2.50 hrs to approximately 9.5 hrs, approximately 2.75hrs to approximately 9.0 hrs, approximately 3.00 hrs to approximately8.5 hrs, approximately 3.25 hrs to approximately 8.0 hrs, approximately3.50 hrs to approximately 7.5 hrs, approximately 3.75 hrs toapproximately 7.0 hrs, approximately 4.00 hrs to approximately 6.5 hrs,approximately 4.25 hrs to approximately 6.0 hrs, approximately 4.50 hrsto approximately 5.5 hrs, approximately 4.75 hrs to approximately 5.0hrs after administering.

In the embodiments of the methods described herein, the sample isilluminated with a wavelength of light selected to give a detectableoptical response, and observed with a means for detecting the opticalresponse. Equipment that is useful for illuminating the dye compounds ofthe invention includes, but is not limited to, tungsten lamps, hand-heldultraviolet lamps, mercury arc lamps, xenon lamps, light emitting diodes(LED), lasers and laser diodes. These illumination sources areoptionally integrated into surgical cameras, laparoscopes andmicroscopes. Preferred embodiments of the invention are dyes that areexcitable at or near the wavelengths 633-636 nm, 647 nm, 660 nm, 680 nmand beyond 700 nm, such as 780 nm, 810 nm and 850 nm as these regionsclosely match the output of relatively inexpensive excitation sources.The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD cameras, video cameras,photographic film.

The NIR imaging probe used was the compound of Formula 1; excitation 773nm and emission 790 nm or independently, Formula 2, excitation 772 nmand emission 787 nm.

The compound of Formula 1 and the compound of Formula 2 as describedherein can be administered in a manner compatible with the dosageformulation, and in such amount as will be effective or suitable for invivo imaging. The quantity to be administered depends on a variety offactors including, e.g., the age, body weight, physical activity, anddiet of the individual, the tissue or organ to be imaged, and type ofprocedure or surgery to be performed. In certain embodiments, the sizeof the dose may also be determined by the existence, nature, and extentof any adverse side effects that accompany the administration of thecompound in a particular individual.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, hereditary characteristics, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and the hostundergoing therapy.

In certain embodiments, the dose may take the form of solid, semi-solid,or lyophilized powder forms, preferably in unit dosage forms suitablefor simple administration of precise dosages. In some embodiments, thedose is provided in a container, vial or syringe at a particular dosagefor one or more administrations.

As used herein, the term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosages for humans and other mammals,each unit containing a predetermined quantity of an imaging agentcalculated to produce the desired onset, tolerability, and/orfluorescent effects, in association with a suitable pharmaceuticalexcipient (e.g., an ampoule). In addition, more concentrated dosageforms may be prepared, from which the more dilute unit dosage forms maythen be produced. The more concentrated dosage forms thus will containsubstantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more times the amount of the imaging agent.

Methods for preparing such dosage forms are known to those skilled inthe art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ED., MackPublishing Co., Easton, Pa. (1990)). The dosage forms typically includea conventional pharmaceutical carrier or excipient and may additionallyinclude other medicinal agents, carriers, adjuvants, diluents, tissuepermeation enhancers, solubilizers, and the like. Appropriate excipientscan be tailored to the particular dosage form and route ofadministration by methods well known in the art (see, e.g., REMINGTON'SPHARMAMCEUTICAL SCIENCES, 18th ED., Mack Publishing Co., Easton, Pa.(1990)).

In certain embodiments, the dosage forms contain a stabilizing agent forthe storage, isolation, purification, and/or lyophilization of thecompounds. Agents for lyophilization include, but are not limited to, asaccharide such as a monosaccharide, a disaccharide or dextran. Othersaccharides include glucose, galactose, xylose, glucuronic acid,trehalose, dextran, hydroxyethyl starch, mannitol, or 5% dextrose.

For parenteral administration, e.g., intravenous injection,intra-arterial injection, subcutaneous injection, intramuscularinjection and the like, the effective dose can be in the form of sterileinjectable solutions and sterile packaged powders. Preferably,injectable solutions are formulated at a pH of from about 4.5 to about7.5, or physiological pH.

In some embodiments, the effective dose for imaging contains alyophilized compound described herein in a high-quality, easilydissolved form which is stable for months at room temperature. Thelyophilized compound of Formula 1 or Formula 2 can be stored in anysuitable type of sealed container, such as a sealed vial or syringe thatcontains an amount of the compound for a single dosage for a subject,such as a human adult. The term “vial” is used broadly herein, to referto any drug-packaging device that is designed and suitable for sealedand sterile storage, shipping, and handling of small (e.g.,single-dosage) quantities of drugs. Single-chamber vials (which wouldcontain only the lyophilized compound, with no water) are well known; atypical single-chamber vial may be designed for use with an intravenousinfusion bag. Alternatively, two-chamber vials can be used that containboth the lyophilized compound and a sterile aqueous solution, to enableimmediate reconstitution and injection of an aqueous liquid containingthe compound of Formula 1 or Formula 2.

Provided herein are kits containing a compound of Formula 1 or acompound of Formula 2. In some embodiments, a kit comprises one or morevials or syringes containing compound of Formula 1 in, for example, alyophilized form. In other embodiments, a kit comprises one or morevials or syringes containing compound of Formula 2 in, for example, alyophilized form. Such kits can also include a pharmaceuticallyacceptable carrier or a sterile aqueous solution, e.g., sterile waterfor reconstituting the compound prior to administration. In some cases,the kit also includes a sterile syringe for parenteral administration ofthe compound or for use with an intravenous infusion bag. The kit canalso include an instruction manual for use.

When ranges are used herein, all combinations and subcombinations ofranges and specific embodiments therein are intended to be included. Useof the term “approximately” when referring to a number or a numericalrange means that the number or numerical range referred to is anapproximation within experimental variability (or within statisticalexperimental error), and thus the number or numerical range may varyfrom, for example, between 1% and 15% of the stated number or numericalrange. The term “comprising” (and related terms such as “comprise” or“comprises” or “having” or “including”) includes those embodiments that“consist of” or “consist essentially of” the described features.

III. Examples Example 1

The compound of Formula 1 may be synthesized by dissolving3,3-Dimethyl-2-[2-[2-chloro-3-[2-[1,3-dihydro-3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-5-sulfo-1-(4-sulfobutyl)-3H-indoliumhydroxide, innersalt, trisodium salt (1 g, 1.05 mmol) in 25 mL of waterand sparging with nitrogen for 15 minutes. Sodium4-hydroxybenzenesulfonate dihydrate (875 mg, 3.77 mmol) was dissolved in3.6 mL 1N NaOH (3.6 mmol) and added to the reaction mixture. Thereaction mixture was placed in an oil bath at 40° C. and stirred for 16hours. The solution was dried by rotary evaporation and the product wasthen recrystallized from 80:20 ethanol:water. The compound was filteredthen washed with ethanol and dried under vacuum at 60° C. for 18 hours.

The polymorph of the compound of Formula 1 (Form A) can be prepared asfollows:

A mixture of3,3-Dimethyl-2-[2-[2-chloro-3-[2-[1,3-dihydro-3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-5-sulfo-1-(4-sulfobutyl)-3H-indoliumhydroxide, innersalt, trisodium salt (20 g, 21 mmol) and Sodium4-hydroxybenzenesulfonate dihydrate (5.84 g, 25.2 mmol) is suspended inwater (120 ml). The suspension is heated to 85° C. where completedissolution is observed. Aqueous sodium hydroxide (10N, 2.5 ml, 25 mmol)is added dropwise and the reaction stirred for 45 min. Isopropanol (360ml) is added slowly to maintain the reaction temperature above 60° C.The mixture is then slowly cooled to ambient temperature and theresulting slurry filtered. The filter cake is rinsed with 40 mlisopropanol:water (3:1) and twice with 40 ml isopropanol and dried at50-60° C. under vacuum to obtain 18.4 g of the compound of Formula 1 asa dark green solid. Ten grams (9 mmol) of this material is thenrecrystallized by dissolving in water (50 ml) and isopropanol (100 ml)at approximately 70° C., and slowly cooling the mixture to ambienttemperature. The solids are collected by filtration and rinsed with 20ml isopropanol:water (2:1) and twice with 20 ml isopropanol and dried at50-60° C. under vacuum to obtain 6.7 g of the compound Formula 1 as acrystalline dark green solid.

X-Ray Powder Diffraction (XRPD) for Form A

XRPD analysis was carried out on a PANalytical X'pert pro, scanning thesamples between 3 and 35° 2θ. The material was gently ground and loadedonto a multiwell plate with Kapton or mylar polymer film to support thesample. The multiwell plate was then loaded into a Panalyticaldiffractometer running in transmission mode and analyzed.

The XRPD data are shown in FIG. 1A-1F. As illustrated, FIGS. 1A and 1Care identical and are both moderately crystalline and represent Form A.FIG. 1B is similar in crystallinity and is Form A.

FIG. 1D is crystalline. FIG. 1E is an enlargment of FIG. 1D. FIG. 1D andFIG. 1E represent Form A.

FIG. 1F is the amorphous form.

Characteristic XRPD angles and d-spacings for the solid state form aresummarized in Table 1 for Form A. Peak positions were measured andtabulated.

TABLE 1 Peak Data of IRDye 800BK Lot: BV736185 (FIG. 1A) XPRD AnalysisPos. Height FWHM d-spacing Rel. Int. [° 2Th.] [cts] [° 2Th.] [Å] [%]4.2384 362.12 0.1279 20.84818 29.2 5.2771 550.29 0.0895 16.74659 44.375.4888 295.59 0.0768 16.10118 23.83 5.9607 293.22 0.0512 14.82759 23.649.5414 221.36 0.1535 9.26957 17.85 10.5728 314.69 0.0768 8.36752 25.3710.9522 224.35 0.1023 8.07854 18.09 11.3914 371.25 0.0768 7.76803 29.9311.9362 322.74 0.0768 7.41466 26.02 12.8323 579.36 0.0895 6.89882 46.7114.1854 785.5 0.1279 6.24366 63.33 15.0743 305.87 0.1279 5.87743 24.6615.3653 384.4 0.1279 5.76676 30.99 15.5532 262.25 0.1023 5.69752 21.1515.8747 444.26 0.0512 5.58284 35.82 16.3152 405.54 0.1535 5.43308 32.716.7216 378.51 0.0768 5.30195 30.52 17.3776 514.77 0.064 5.10327 41.5118.2698 226.8 0.1023 4.856 18.29 19.1518 465.59 0.1535 4.63431 37.5419.5492 393.65 0.1279 4.54099 31.74 20.1805 277.83 0.1023 4.40035 22.420.7531 1146.9 0.1151 4.2802 92.47 21.2128 1240.24 0.1151 4.18848 10021.8521 229.87 0.2047 4.06738 18.53 22.6123 239.17 0.1535 3.93232 19.2823.389 331.71 0.064 3.80346 26.75 23.8748 148.63 0.1535 3.72716 11.9824.3664 124.78 0.1535 3.65307 10.06 24.9407 221.2 0.1279 3.57024 17.8426.4158 297.98 0.1279 3.37412 24.03 28.658 136.22 0.1791 3.11503 10.98

Characteristic XRPD angles and d-spacings for the solid state form aresummarized in Table 2 for Form A. Peak positions were measured andtabulated.

TABLE 2 Peak data of IRDye 800BK Lot: VE- 759-24-1 (FIG. 1E) XRPDAnalysis Pos. Height FWHM d-spacing Rel. Int. [° 2Th.] [cts] [° 2Th.][Å] [%] 4.272 1845.05 0.064 20.68408 100 9.6191 863.9 0.0895 9.1949146.82 10.5611 267.84 0.1023 8.37677 14.52 12.4823 217.24 0.0768 7.0914511.77 12.7274 748.04 0.0895 6.95545 40.54 12.93 1108.7 0.0768 6.8469260.09 13.4567 353.68 0.1279 6.58007 19.17 14.197 188.01 0.1023 6.2386110.19 15.4372 537.33 0.064 5.74009 29.12 15.8734 703.35 0.0384 5.5832938.12 16.1055 637.51 0.064 5.50336 34.55 16.6046 222 0.1279 5.3390612.03 17.1929 293.61 0.1023 5.15766 15.91 17.559 419.11 0.1023 5.0509422.72 18.0101 705.32 0.1023 4.92543 38.23 18.2864 1035.04 0.1663 4.8516356.1 18.7831 635.82 0.0512 4.72445 34.46 19.1781 617.24 0.0895 4.6280133.45 19.4323 420.97 0.4093 4.56805 22.82 19.7836 680.51 0.064 4.4877236.88 20.2392 744.22 0.0512 4.38771 40.34 20.5039 801.47 0.1151 4.3316643.44 20.8076 960.22 0.1407 4.26912 52.04 21.1497 546.41 0.1791 4.2008329.61 21.7763 541.2 0.064 4.08136 29.33 22.5703 573.98 0.0512 3.9395431.11 23.1948 292.69 0.1023 3.83487 15.86 23.6245 311.01 0.1279 3.7660816.86 24.6218 243.08 0.1535 3.61575 13.17 25.2623 343.69 0.1791 3.5255118.63 26.3234 274.93 0.1791 3.38575 14.9 28.0536 225.6 0.1535 3.1807512.23 28.6694 301.22 0.2047 3.11382 16.33

In one embodiment, it is possible to make Form B from Form A. Form A iscrystalline and exists as small birefringent particles with no definedmorphology. The material is hygroscopic (27 wt % uptake up-to 90% RH)and can change form under stress conditions. A form change does occurwhen Form A is subjected to 40° C./75% RH. This form corresponds withthe form found with the post-GVS sample. HPLC purity showed no change.

FIG. 1H and FIG. 1I represent Form B. For comparison, FIG. 1G isidentical to FIG. 1D.

Characteristic XRPD angles and d-spacings for the solid state form aresummarized in Table 3 for Form B. Peak positions were measured andtabulated.

TABLE 3 Peak data of IRDye 800BK Lot: VE- 759-24-1 Form B XRPD AnalysisPos. Height FWHM d-spacing Rel. Int. [° 2Th.] [cts] [° 2Th.] [Å] [%]5.2989 1193.55 0.064 16.67783 52.81 5.9816 649.44 0.0512 14.77594 28.749.535 387.23 0.064 9.27578 17.13 10.6007 690.31 0.1023 8.3456 30.5510.9387 376.49 0.0895 8.08848 16.66 11.3924 413.39 0.064 7.76734 18.2911.9715 817.54 0.0768 7.39285 36.18 12.8677 587.96 0.1279 6.8799 26.0214.2153 1273.22 0.0936 6.22544 56.34 14.2827 1000.8 0.078 6.21161 44.2815.9468 606.88 0.2496 5.55317 26.85 16.3781 600.12 0.1092 5.4079 26.5516.7597 562.34 0.1092 5.28561 24.88 17.3714 797.24 0.078 5.10083 35.2817.7698 277.37 0.1872 4.98736 12.27 18.4312 246.4 0.156 4.80986 10.918.6816 475.06 0.1872 4.74595 21.02 19.1392 766.24 0.078 4.63351 33.9119.5987 600.29 0.0936 4.52589 26.56 20.1626 509.11 0.0624 4.40057 22.5320.7073 1255.16 0.1404 4.28603 55.54 21.2464 2259.95 0.1092 4.17848 10021.8382 456.8 0.156 4.06656 20.21 22.6653 385.85 0.1872 3.92 17.0723.4295 644.04 0.2808 3.79383 28.5 23.9381 351.56 0.1872 3.71438 15.5624.9551 451.86 0.2184 3.56525 19.99 26.4745 574.6 0.0468 3.36399 25.43

Example 2

The compound of Formula 2 may be synthesized by dissolving3,3-Dimethyl-2-[2-[2-chloro-3-[2-[1,3-dihydro-3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-5-sulfo-1-(4-sulfobutyl)-3H-indoliumhydroxide, innersalt, trisodium salt (1 g, 1.05 mmol) in 25 mL of waterand sparging with nitrogen for 15 minutes. Sodium phenoxide trihydrate(390 mg, 2.29 mmol) was dissolved in 2 mL ultrapure water and added tothe reaction mixture. The reaction mixture was placed in an oil bath at40° C. and stirred for 4 hours. The solution was dried by rotaryevaporation and the product was then recrystallized from 90:10ethanol:water. The compound was filtered then washed with ethanol anddried under vacuum at 60° C. for 18 hours.

Comparative Example 1

The structure of the compound of comparative example 1, also referred toas 800CW, is shown below:

The structure of indocyanine green (ICG) dye is shown below:

Example 3: In Vitro

In vitro cell-based assays with two carcinoma cell lines (A431 and A549)were done to assess non-specific binding of the compound of Formula 1and the compound of Formula 2. The dyes were compared to 800CW. This isshown in FIGS. 2A, 2B, and 2C.

The compound of Formula 2 demonstrated a significant increase innon-specific binding compared to either the compound of Formula 1 and800CW by two-fold. The compound of Formula 1 was more comparable to800CW.

To confirm all three compounds have comparable fluorescence, a dilutionseries was made and imaged on an Odyssey SA fluorescence imaging system(LI-COR Biosciences). The fluorescence in each well was quantified withthe instrument software. FIG. 2C shows that all three compounds in thistest were very close in fluorescence, thus confirming that the increasedfluorescence at higher concentrations noted in plate-based assay islikely due to a tendency of the compound to bind non-specifically tocells.

In general, low non-specific binding is an advantage. Both cell linesshow low non-specific binding for all compounds at concentrations below1 μM. However all begin to show some binding at higher concentrations.

Example 4: In Vivo

Animals injected with the probes were imaged over time using the 800fluorescence channel of the Pearl Impulse Imaging System (LI-CORBiosciences). Clearance profiles for 800CW, the compound of Formula 1,and the compound of Formula 2 were carried out in nude mice (dose of 1nmole for all compounds). Serial images and excised organs wereevaluated over a 24 hr period. Dorsal image series are presented in FIG.3. A Pearl Impulse small animal imaging system was used for all animaland organ image acquisitions.

The compound of comparative example 1 is used as the reference. From theDorsal view there is no kidney signal for any probe. At 2 hrs postinjection, the compound of Formula 2 exhibits high signal, emanating inthe liver region visible from the back. The compound of Formula 2 neverfully disseminates because it is collected rapidly in the liver and by 5hr post injection, the whole body signal is low compared to 800CW andthe compound of Formula 1.

Example 5: Ventral Images

Ventral images are presented in FIG. 4. From visual examination, thecompound of Formula 1 and 800CW are similar in their clearance pattern.These two probes appear to clear rapidly via the kidney. This feature isvery useful for imaging the urinary system.

The compound of Formula 2, on the other hand, clears through the liver.This feature is a very useful attribute for a biliary duct monitoringdye which may “mimic” ICG that is currently used for this clinicalapplication. From the in vitro plate-based assays it appeared both thecompound of Formula 1 and the compound of Formula 2 have similarfluorescence intensities to 800CW. However, from the in vivo data, itappeared that the compound of Formula 1 moves rapidly through thetissues but is retained for an extended period in organs.

Bladder signal is the strongest signal noted for 800CW and the compoundof Formula 1 probes (purple arrows).

Whole body fluorescence numbers for the three probes (ventral view) areshown in FIG. 5. Non-invasive imaging will not capture signals for thefull depth of the animal but the intensity values are useful to show arelative trend.

There is a slower clearance for 800CW and the compound of Formula 1 ascompared to the compound of Formula 2. The signal for the compound ofFormula 2 appears to be lower overall than the signal for 800CW and thecompound of Formula 1, however, it appears to rapidly collect in theliver. At 24 hrs post administration, the remaining fluorescence isdiminished for all three probes to roughly the same extent.

Another way of examining fluorescence signals is by measuring the % ofthe compound remaining at a particular time point. The 30 min time pointis the baseline to which other time points are normalized. This data arepresented below in Table 4.

30 120 300 1440 min min min min Compound of comparative example 1 100 8523 0.8 (800CW) Compound of Formula 1 100 81 19 1.4 (800BK-sulfonate)Compound of Formula 2 100 70 14 1.4 (800BK-NOS)

The percent signal remaining at each time is shown with the 30 min timepoint post-injection, which is used as the baseline.

The greatest reduction occurs for the compound of Formula 2 at 2 hrs.The compound of Formula 1 is very similar to 800CW and by 24 hrs postinjection, all compounds are very similar in remaining signals at lessthan 2% of baseline.

Example 5: Organs

Organs were excised at 24 hrs and imaged. Data is presented in FIG. 6.

The compound of Formula 1 is retained at higher levels in the liver andkidney compared to the compound of Formula 2 at 24 hrs post injection.Surprisingly, the compound of Formula 2, which clears predominantly fromthe liver, has lower liver signals at 24 hrs when compared to 800CW andthe compound of Formula 1. This may be explained by a slower overallclearance from tissue for the compound of Formula 1 as compared to 800CWand the compound of Formula 2.

None of 800CW or the compound of Formula 1 or the compound of Formula 2is detected in the brain and is likely due to their inability to crossthe blood brain barrier.

FIG. 7 illustrates the ureters along with kidney and bladder. When thecompound of Formula 1 was administered to screen the ureter of thekidney, an image of the region was captured post-administration.

FIG. 8 illustrates fluorescence images of stained electrophoresis gelsof selected proteins showing non-specific or affinity of the namedcompounds to human (H), bovine (B), ovalbumin (O), or 5% FBS (fetalbovine serum). It is clear that the compound of Formula 2 has potentialto be useful for biliary duct examinations, similar to ICG.

Example 7: Mouse Urine

With the primary elimination route being renal for the compound ofFormula 1, the urine output in mice was analyzed. Urine was obtainedfrom mice that received: neither of the compound of Formula 1 nor thecompound of Formula 2 (control), the compound of Formula 1, ICG, and thecompound of Formula 2. The urine was extracted with anacetonitrile:methanol mix and imaged on an Odyssey CLx imager (LI-CORBiosciences) as shown in FIG. 9.

No fluorescent signal was seen for the control and ICG. ICG iseliminated by the liver so no signal is expected. There was a slightfluorescence for the compound of Formula 2 that suggests liver excretionis not quite exclusive.

Example 8: Comparison with ICG

A comparison between the compound of Formula 2 and ICG was conducted fortheir common application in imaging the biliary duct (FIGS. 10A and10B). A Pearl Impulse small animal imaging system was used for allanimal and organ image acquisitions. ICG is not very soluble in aqueousmedia and gives a much weaker fluorescent signal than the compound ofFormula 2 in vivo. A 50 nmole solution of ICG in phosphate buffer salinesolution was prepared, injected intravenously, and compared forlocalization with a 1 nmole solution of the compound of Formula 2 attime periods of 1 min, 30 min, 2 hrs, 5 hrs, and 24 hrs.

A comparison of the dose responses for the compound of Formula 2 areshown in FIG. 10B (1 nmole), FIG. 10C (0.5 nmole), and FIGS. 10D and 10E(0.1 nmole) at time periods of 1 min, 30 min, 2 hrs, 5 hrs, and 24 hrs.All images are presented using the same LUT table except FIG. 10E wherethe LUT for FIG. 10D has been reduced to brighten the 800 nm signal sothey are visible.

The gall bladder is visible by 30 min post-intravenous administrationfor the 1 nmole (FIG. 10B) and 0.5 nmole (FIG. 10C) doses and continuesto be visible in the 2 hrs and 5 hrs images. The images for the 0.1nmole dose in FIG. 10D appear to show very low detection signal usingthe same scale as the 1 nmole and 0.5 nmole dose series. However, if thescale is adjusted as in FIG. 10E, the gall bladder target region isvisible on a very low background. A wide range of dosing is possible toaccommodate a particular imaging system or application.

Example 9: Excised Organ Evaluation

Fluorescent signal intensities of various organs were examined at 24 hrsand 72 hrs for the compound of Formula 1 and the compound of Formula 2(FIG. 11A-11E). Organs examined included: heart (Ht), lungs (Ln), kidney(Kd), liver (Lv), spleen (Spl), intestine (Int), brain (Br), and muscle(Ms). At 24 hrs, there remains some signal in the liver and kidney butby 72 hrs the signal is diminished substantially.

FIG. 11A shows the results using the control (800CW), 24 hours postadministration.

FIG. 11B shows the results using a compound of Formula 2, 24 hours postadministration. FIG. 11C shows the results using a compound of Formula1, 24 hours post administration.

FIG. 11D-11E show the results 72 hours post administration of Formula 2and Formula 1, respectively.

FIG. 11F shows a surgically opened animal for determining whetherbiliary structures are visible. The animal's liver was positioned toexpose the critical structures. The image series of FIG. 11F-11H presentthe white light image of the liver with lobes raised to expose the gallbladder and biliary duct associated with Calot's triangle; the 800 nmimage as detected by fluorescence studies of a compound of Formula 2;and a composite image overlaying the 800 nm image atop the white lightimage.

Example 10: Elimination Routes in Mice

This example describes an experiment to evaluate the excretion of thedyes described herein from animals injected with such. The dyes testedwere stable cyanine dyes (IRdyes): 800CW, 800BK-sulfonate (compound ofFormula 1; 800BK or BK) and 800BK-NOS (compound of Formula 2; 800NOS orNOS). In this study 12 nude mice (3 per treatment group) were injectedwith either (1) no probe (control group); (2) 800CW; (3) 800NOS; or (4)800BK. The three probes (800CW-1166 g/mole (should have been 1091.1g/mole), 800NOS-1011.09 g/mole, and 800BK-1113.14 g/mole) were dissolvedin PBS. A spot test of the three probes was performed to detect thefluorescent signal when diluted to the injection dose of 1 nmole/100 μl(FIG. 12). The fluorescence of 800CW was slightly higher than that of800NOS and 800BK.

The mice received 1 nmole of dye by tail vein injection. The mice wereimaged serially over 24 hours after IV injection: 5 minutes, 1 hour, 2hours, 4 hours, 6 hours and 24 hours after injection. A representativeseries of images are presented in FIGS. 13A-13C of one animal per dye.All the data was normalized to the same LUT. A control mouse (no probe)was used as a reference (no signal control). Imaging was performed usingthe Pearl® Trilogy Imaging System (LI-COR®).

The mice were then sacrificed and their organs and tissue (i.e., liver,kidney, lungs, spleen and muscle) were harvested. The organs were imagedto detect fluorescence. When the same LUT was used for the animal andorgan images, fluorescence in the organs was not detectable. As such,the low end of the animal scale was expanded such that the images of theorgans could be evaluated (FIG. 14). For example, the “Organs 1/10^(th)scale” was used for the images of FIGS. 15A-15D and the “Organs1/100^(th) scales was used for the images of FIGS. 16A-16D.

FIG. 15A shows no fluorescence in the organs of the control animal.Kidneys (lower left corner) and liver (lower right corner) had adetectable signal in the animals treated with 800CW (FIG. 15B), 800NOS(FIG. 15C), and 800BK (FIG. 15D). No muscle (top right corner) or lungtissue (top left corner) was visible in the treatments except for the800CW treatment. At the 1/10^(th) LUT scale, the high level is 1/10^(th)that of the whole animal LUT scale. The data shows that there is onlyresidual dye remaining in these organs when imaging at the whole animalLUT scale.

FIGS. 16A-D show some signal in the organs if the whole animal LUT scaleis reduced to 100× such that the adjusted scale is 1/100^(th) the wholeanimal scale. FIG. 16A shows the signal from the control animal. FIGS.16B, 16C and 16D shows the signal from organs of the 800CW treatedanimal, the 800NOS treated animal and the 800BK treated animal,respectively.

800CW and 800BK was excreted renally. The higher overall signal in the800CW animals is mainly due to the accidentally higher concentration ofthe probe injected into the animals.

800NOS was eliminated from the body via the biliary tract (liver, gallbladder and bile ducts) and intestines. In the 800NOS treated animals,the liver was visible at 15 minutes and then the gall bladder, as thedye rapidly left the liver. The data also showed that the dye was movedinto the intestines for excretion. The predominant signal between 2-6hours was in the intestines. At 24 hours post injection the intestinalsignal was barely detectable.

The organs were imaged under three LUT scales; each being progressivelysmaller in the signal range covered. The lower levels of eachprogressive scale were expanded to cover the full red to blue colorrange (FIG. 14). Very little signal remained in any of the target organssuch as liver, kidney, lungs and muscle. Similar signal intensities werefound in the kidney for 800CW and 800BK. In the liver, 800BK had ahigher signal compared to 800CW. The results show that 800BK has alonger retention time in the liver compared to 800CW and 800NOS. Thismay be due to increased (higher) plasma protein binding of 800BK than800CW.

This example shows that the clearance of IRDye 800NOS is rapid from thewhole body to the biliay system with a very short retention time in theliver and also rapid excretion into the intestines. The rapid clearanceby the liver implies that 800NOS has low plasma protein bindingactivity.

Example 11: Ureter Visualization During a Hysterectomy

The pharmaceutical formulation of Formula 1 is dissolved in a vial (25mg) with 5 mL of saline (0.9% sodium chloride) and is administered to apatient via a bolus injection at the concentration of 5 mg/mL, 15minutes prior to the surgery. The medical device for this procedure isthe PINPOINT Endoscopic Fluorescence Imaging System (Novadaq,Mississauga, Ontario, Canada). At any point during the procedure, whenthe surgeon needs to identify the ureter, the device's mode is switchedto the near-infrared detection imaging, and the surgeon visualizes theureter via the monitor or display of the medical device. Thisidentification of the ureter is visualized on the monitor as an overlayimage, in which the surgeon can simultaneously locate the ureter withwhite light imaging.

Example 12: Biliary Duct Visualization During a Cholecystectomy

The pharmaceutical formulation of Formula 2 is dissolved in a vial (25mg) with 5 mL of saline (0.9% sodium chloride) and is administered to apatient via a bolus injection at the concentration of 5 mg/mL, 15minutes prior to the surgery starting. The medical device for thisprocedure is the da Vinci Firefly Surgical System (Intuitive Surgical,Sunnyvale Calif.). During the procedure, when the surgeon needs toidentify the biliary duct, the device's mode is switched to thenear-infrared detection imaging, and the surgeon identifies the biliaryduct via the monitor or display of the medical device by switchingbetween the white light image and the near-infrared image as needed.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method for kidney ureter imaging in a subject,the method comprising: providing a polymorphic compound of sodium2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonateof Formula 1:

wherein the polymorph is a member selected from the group consisting ofForm A having an X-ray powder diffraction pattern comprising peaks at2-theta at 4.30±0.2°, 9.60±0.2° and 12.9°±0.2° and Form B having anX-ray powder diffraction pattern comprising peaks at 2-theta at5.3°±0.2°, 14.2°±0.2° and 21.2°±0.2°; admixing said polymorphic compoundalong with a saccharide stabilizing agent and thereafter dissolving in apharmaceutically acceptable carrier to make a pharmaceuticalcomposition; administering the pharmaceutical composition to thesubject; exposing tissue of the subject's renal system toelectromagnetic radiation; and detecting fluorescence radiation from thecompound.
 2. The method of claim 1, wherein the polymorphic form is FormA.
 3. The method of claim 1, wherein the polymorphic form is Form B. 4.The method of claim 1, wherein the saccharide stabilizing agent is amember selected from the group consisting of a monosaccharide, adisaccharide and dextran.
 5. The method of claim 4, wherein thesaccharide stabilizing agent is a member selected from the groupconsisting of glucose, galactose, xylose, glucuronic acid, trehalose,hydroxyethyl starch, mannitol, and 5% dextrose.
 6. The method of claim1, wherein the pharmaceutical carrier is selected from the groupconsisting of physiological sterile saline solution, sterile watersolution, pyrogen-free water solution, isotonic saline solution, andphosphate buffer solution.
 7. The method of claim 1, wherein theadministering is conducted at a diagnostic effective amount of thecompound ranging between approximately 30.0 μg/kg and approximately3000.0 μg/kg.
 8. The method of claim 1, wherein the administering isconducted at a diagnostic effective amount of the compound rangingbetween approximately 35.0 μg/kg and approximately 600.0 μg/kg.
 9. Themethod of claim 1, wherein the administering is conducted at adiagnostic effective amount of the compound ranging betweenapproximately 40.0 μg/kg and approximately 420.0 μg/kg.
 10. The methodof claim 1, wherein the administering is conducted at a diagnosticeffective amount of the compound ranging between approximately 60.0μg/kg and approximately 120.0 μg/kg.
 11. The method of claim 1, whereinthe polymorphic form and saccharide stabilizing agent are provided in alyophilized form and reconstituted in the pharmaceutical carrier beforeadministering to the subject.
 12. The method of claim 1, wherein theadministering is conducted intravenously.
 13. The method of claim 1,further comprising measuring a fluorescence intensity of theadministered compound remaining at the tissue of the subject's renalsystem at a time period after administering.
 14. The method of claim 13,wherein the measured fluorescence intensity of the administered compoundis higher in the kidney as compared to a measured fluorescence intensityof the administered compound in one or more of the spleen, intestine,heart, lungs, muscle, or combinations thereof approximately up to sixhours after administering.
 15. The method of claim 13, wherein themeasured fluorescence intensity of the administered compound isbackground fluorescence approximately 24 hours after administering. 16.The method of claim 1, wherein the kidney ureter imaging is performedduring a procedure selected from the group consisting of a laparoscopicprocedure, a robotic procedure, a robotic laparoscopic procedure, and anopen procedure.
 17. The method of claim 13, wherein the measuredfluorescence intensity of the administered compound is measured lessthan 5.0 hrs, 4.75 hrs, 4.50 hrs, 4.25 hrs, 4.00 hrs, 3.75 hrs, 3.50hrs, 3.25 hrs, 3.00 hrs, 2.75 hrs, 2.50 hrs, 2.25 hrs, 2.00 hrs, 1.75hrs, 1.50 hrs, 1.25 hrs, 1.00 hrs, 0.90 hrs, 0.80 hrs, 0.70 hrs, 0.60hrs, 0.50 hrs, 0.40 hrs, 0.30 hrs, 0.20 hrs after administering thecompound.